extra HpaII site in pEGFP-N1's MCS???

Paul Kitts kitts at ncbi.nlm.nih.gov
Wed Jul 19 13:28:57 EST 2000


Arno,
It sounds like you may actually have pEGFP-N2 and not N1:
               AgeI
              ------
pEGFP-N1      ACCGGT
pEGFP-N2      ACCGGCCGGT
               --------
              HpaIIHpaII

-=Paul=-

Paul Kitts
National Center for Biotechnology Information 
NIH/NLM

----------------------------------------------------------------------
Dear fluorpro's, 

After creating a construct for the fusion of a keratinocyte protein to 
the N-terminus of EGFP
(pEGFP-N1) and going through several unsuccesful attempts to generate 
a fluorescent signal, we found
by means of sequence analysis (both on DNA level and RNA level 
(RT-PCR, 5' and 3'RACE)) there was
an additional HpaII site (CCGG) at MCS position 670 (next to Age I 
position). 

Has anyone in anyway come about these 4 extra nucleotides or heared 
about their presence in
pEGFP-N1? 

N.B. The construct was supposed to have a "linker" of 21 nucleotides 
of the MCS followed by the ATG of
EGFP. The extra 4 nucleotides are not in a region where digestions 
and/or ligations were performed. 

greetings, 

Arno Pol 
dept. of Dermatology, 
University Medical Centre Nijmegen, 
the Netherlands. 


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